scholarly journals POLYCLONAL ANTIBODIES AGAINST HUMAN PLASMINOGEN: PURIFICATION, CHARACTERIZATION AND APPLICATION

2020 ◽  
Vol 13 (6) ◽  
pp. 50-57
Author(s):  
T. A. Yatsenko ◽  
Keyword(s):  
Flow Cytometry ◽  
Polyclonal Antibodies ◽  
Blood Collection ◽  
Salting Out ◽  
Cell Functions ◽  
Isolation And Characterization ◽  
Wide Range ◽  
Direct Elisa ◽  
Human Plasminogen

The plasminogen/plasmin system plays a crucial role in fibrinolysis and regulation of cell functions in a wide range of normal and pathological processes. Investigation of plasminogen/plasmin functions requires the availability of well-characterized and effective molecular tools, such as antibodies. In the present work, the isolation and characterization of rabbit polyclonal antibodies against human plasminogen are described and approaches for the identification of plasminogen and its fragments using the purified antibodies are demonstrated. For the antibodies isolation, standard animal immunization and blood collection procedures, serum isolation, protein salting out and affinity chromatography were performed. For the antibodies characterization and application, the following methods were used: enzyme linked immunoassay (ELISA), Western blotting, FITC-protein conjugation, flow cytometry and spectrofluorometry. The obtained polyclonal rabbit anti-human plasminogen antibodies interacted with human Glu- and Lys-plasminogen, kringles 1-3 and 1-4 of plasminogen, mini-plasminogen, the heavy and light chain of plasmin. We propose the application of anti-plasminogen antibodies for the direct ELISA, Western blot analysis, and for flow cytometry and spectrofluorometric analysis of plasminogen binding with cells. The obtained anti-plasminogen antibodies are promising tools for the investigation of plasminogen/plasmin system functions, either fibrinolytic or signaling.

scholarly journals Isolation and Characterization of Strain Exiguobacterium sp. KRL4, a Producer of Bioactive Secondary Metabolites from a Tibetan Glacier

2021 ◽  
Vol 9 (5) ◽  
pp. 890
Author(s):  
Pietro Tedesco ◽  
Fortunato Palma Esposito ◽  
Antonio Masino ◽  
Giovanni Andrea Vitale ◽  
Emiliana Tortorella ◽  
...  
Keyword(s):  
Phenotypic Analysis ◽  
Biological Assays ◽  
C Elegans ◽  
Isolation And Characterization ◽  
Wide Range ◽  
Protein Encoding ◽  
Nematocidal Activity ◽  
Extremophilic Microorganisms ◽  
Unique Source

Extremophilic microorganisms represent a unique source of novel natural products. Among them, cold adapted bacteria and particularly alpine microorganisms are still underexplored. Here, we describe the isolation and characterization of a novel Gram-positive, aerobic rod-shaped alpine bacterium (KRL4), isolated from sediments from the Karuola glacier in Tibet, China. Complete phenotypic analysis was performed revealing the great adaptability of the strain to a wide range of temperatures (5–40 °C), pHs (5.5–8.5), and salinities (0–15% w/v NaCl). Genome sequencing identified KRL4 as a member of the placeholder genus Exiguobacterium_A and annotation revealed that only half of the protein-encoding genes (1522 of 3079) could be assigned a putative function. An analysis of the secondary metabolite clusters revealed the presence of two uncharacterized phytoene synthase containing pathways and a novel siderophore pathway. Biological assays confirmed that the strain produces molecules with antioxidant and siderophore activities. Furthermore, intracellular extracts showed nematocidal activity towards C. elegans, suggesting that strain KRL4 is a source of anthelmintic compounds.

scholarly journals Isolation and Characterization of Bacteriophage ZCSE6 against Salmonella spp.: Phage Application in Milk

Biologics ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 164-176
Author(s):  
Abdallah S. Abdelsattar ◽  
Anan Safwat ◽  
Rana Nofal ◽  
Amera Elsayed ◽  
Salsabil Makky ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Raw Milk ◽  
Salmonella Spp ◽  
Isolation And Characterization ◽  
Food Borne ◽  
Wide Range ◽  
Almost All ◽  
And Control ◽  
Milk And Dairy Products

Food safety is very important in the food industry as most pathogenic bacteria can cause food-borne diseases and negatively affect public health. In the milk industry, contamination with Salmonella has always been a challenge, but the risks have dramatically increased as almost all bacteria now show resistance to a wide range of commercial antibiotics. This study aimed to isolate a bacteriophage to be used as a bactericidal agent against Salmonella in milk and dairy products. Here, phage ZCSE6 has been isolated from raw milk sample sand molecularly and chemically characterized. At different multiplicities of infection (MOIs) of 0.1, 0.01, and 0.001, the phage–Salmonella interaction was studied for 6 h at 37 °C and 24 h at 8 °C. In addition, ZCSE6 was tested against Salmonella contamination in milk to examine its lytic activity for 3 h at 37 °C. The results showed that ZCSE6 has a small genome size (<48.5 kbp) and belongs to the Siphovirus family. Phage ZCSE6 revealed a high thermal and pH stability at various conditions that mimic milk manufacturing and supply chain conditions. It also demonstrated a significant reduction in Salmonella concentration in media at various MOIs, with higher bacterial eradication at higher MOI. Moreover, it significantly reduced Salmonella growth (MOI 1) in milk, manifesting a 1000-fold decrease in bacteria concentration following 3 h incubation at 37 °C. The results highlighted the strong ability of ZCSE6 to kill Salmonella and control its growth in milk. Thus, ZCSE6 is recommended as a biocontrol agent in milk to limit bacterial growth and increase the milk shelf-life.

Paucimyces polynucleatus gen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the faeces of a wild blackbuck antelope

2021 ◽  
Vol 71 (6) ◽  
Author(s):  
Radwa A. Hanafy ◽  
Noha H. Youssef ◽  
Mostafa S. Elshahed
Keyword(s):  
Ribosomal Subunit ◽  
New Genus And Species ◽  
Isolation And Characterization ◽  
Faecal Samples ◽  
Wide Range ◽  
Culture Independent ◽  
Antilope Cervicapra ◽  
Spherical Vesicles ◽  
Sequence Similarities

The anaerobic gut fungi (AGF; phylum Neocallimastigomycota) reside in the alimentary tracts of herbivores. Multiple novel, yet-uncultured AGF taxa have recently been identified in culture-independent diversity surveys. Here, we report on the isolation and characterization of the first representative of the RH5 lineage from faecal samples of a wild blackbuck (Indian Antelope, Antilope cervicapra) from Sutton County, Texas, USA. The isolates displayed medium sized (2–4 mm) compact circular colonies on agar roll tubes and thin loose biofilm-like growth in liquid medium. Microscopic examination revealed monoflagellated zoospores and polycentric thalli with highly branched nucleated filamentous rhizomycelium, a growth pattern encountered in a minority of described AGF genera so far. The obtained isolates are characterized by formation of spherical vesicles at the hyphal tips from which multiple sporangia formed either directly on the spherical vesicles or at the end of sporangiophores. Phylogenetic analysis using the D1/D2 regions of the large ribosomal subunit (D1/D2 LSU) and the ribosomal internal transcribed spacer 1 (ITS1) revealed sequence similarities of 93.5 and 81.3%, respectively, to the closest cultured relatives (Orpinomyces joyonii strain D3A (D1/D2 LSU) and Joblinomyces apicalis strain GFH681 (ITS1). Substrate utilization experiments using the type strain (BB-3T) demonstrated growth capabilities on a wide range of mono-, oligo- and polysaccharides, including glucose, xylose, mannose, fructose, cellobiose, sucrose, maltose, trehalose, lactose, cellulose, xylan, starch and raffinose. We propose accommodating these novel isolates in a new genus and species, for which the name Paucimyces polynucleatus gen. nov., sp. nov. is proposed.

23 The isolation and characterization of the gene for human plasminogen

1988 ◽  
Vol 2 ◽  
pp. 11
Author(s):  
M.R. Martzen ◽  
T.E. Petersen ◽  
A. Ichinose ◽  
E.W. Davie
Keyword(s):  
Isolation And Characterization ◽  
Human Plasminogen

Dicoumarol-induced prothrombins containing 6, 7, and 8 γ-carboxyglutamic acid residues: isolation and characterization

1989 ◽  
Vol 67 (8) ◽  
pp. 411-421 ◽  
Author(s):  
Om P. Malhotra
Keyword(s):  
Gel Electrophoresis ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Calcium Dependent ◽  
Isolation And Characterization ◽  
Carboxyglutamic Acid ◽  
K Deficiency ◽  
Agar Gel Electrophoresis

Isolation and characterization of γ-carboxyglutamic acid (Gla) deficient prothrombins induced by Warfarin or dicoumarol are useful for studying the role of specific Gla residues in prothrombin. In addition to 7-Gla prothrombin, we have isolated two more atypical prothrombins from the barium citrate eluate, one containing 6.11, and the other, 7.85 Gla residues, presumably 6- and 8-Gla prothrombins. The actual Gla content of the 7-Gla isomer was 7.05. Each of the 6-, 7-, and 8-Gla variants showed a single component by agar or dodecyl sulfate Polyacrylamide gel electrophoresis. When agar gel electrophoresis was performed in calcium, each of the variants moved more rapidly than normal (10-Gla) prothrombin. In the presence of EDTA, the 8-Gla isomer exhibited the fastest mobility, equivalent to that of normal prothrombin, followed by 7-, and then 6-Gla variants. The physiological activities of the isomers were found to be 18 to 23% for 8-, 6 to 8% for 7-, and 2 to 3% of normal prothrombin for 6-Gla variant. Prothrombin fragment 1, derived from 8-Gla prothrombin, exhibited 23% of calcium-induced fluorescence quenching, compared with 40% for 10-Gla and 8% or less for 7- and 6-Gla fragments 1. Competition radioimmunoassay data show that calcium-dependent anti (normal) prothrombin polyclonal antibodies are not specific for 10-Gla prothrombin, since the 7- and 8-Gla isomers were able to displace radiolabeled (125I) normal prothrombin.Key words: prothrombin, blood clotting, dicoumarol, Warfarin, γ-carboxyglutamic acid, vitamin K deficiency.

Isolation and Characterization of CD4+ T Cells Specific for the HPA 1a Epitope Associated with Neonatal Alloimmune Thrombocytopenia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2091-2091
Author(s):  
Maria T. Ahlen ◽  
Mette K. Killie ◽  
Bjorn Skogen ◽  
Anne Husebekk ◽  
Tor B. Stuge
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cell Detection ◽  
Severe Thrombocytopenia ◽  
Isolation And Characterization ◽  
T Cell Lines ◽  
Alloimmune Thrombocytopenia

Abstract Neonatal alloimmune thrombocytopenia (NAIT) can cause severe complications such as intrauterine death or intracranial hemorrhage (ICH) in the newborn, and is caused by the transfer of platelet-depleting antibodies from the mother to the fetus during pregnancy. These antibodies react with allogeneic epitopes, most commonly human platelet antigen (HPA) 1a, when present on fetal platelets. Although these responses are thought to be a result of a T cell-dependent immune response, HPA 1a specific T cells have not yet been isolated. To examine whether HPA 1a specific T cells could be detected and isolated, we collected PBMC post delivery from an HPA 1a negative mother who gave birth to an HPA 1a positive neonate suffering from severe thrombocytopenia (platelet count &lt;50×109/L). The cells were stimulated with HPA 1a peptides (20aa) in long term cultures supplemented with IL-7 and IL-2, and subsequently, IL-15. After 4 weeks in culture these cells were labeled with CFSE dye and restimulated with HPA 1a or control peptides. After additional 2 weeks in culture supplemented with IL-2 and IL-15, specific proliferative responses were detectable by CFSE dye dilution by flow cytometry. The cells were cloned by fluorescent-activated cell sorting (FACS) and expanded in numbers with anti-CD3 stimulation in the presence of irradiated allogeneic PBMC and IL-2. The resulting clonal T cell lines were characterized in proliferation assays, ELISPOT assays and phenotyped by flow cytometry. All clones were CD3+, CD4+ and CD19−, and the majority of the clones proliferated and secreted cytokines in response to stimulation with HPA 1a peptides, but not control peptides. In ELISPOT assays, peptide-pulsed antigen-presenting cells were required for T cell detection. These clonal HPA 1a specific CD4+ T cell lines represent formal evidence of the existence of HPA 1a specific T cell responses related to NAIT and will serve as important tools for further characterization of maternal immune responses associated with NAIT.

scholarly journals Isolation and characterization of omnipotent suppressors in the yeast Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 124 (3) ◽  
pp. 515-522
Author(s):  
L P Wakem ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Low Temperatures ◽  
Carbon Sources ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hypertonic Medium ◽  
Isolation And Characterization ◽  
Wide Range ◽  
Chromosome Ii

Abstract Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the psi+ extrachromosomal determinant. The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers. The suppressors were further distinguished by differences in growth on paromomycin medium, hypertonic medium, low temperatures (10 degrees), nonfermentable carbon sources, alpha-aminoadipic acid medium, and by their dominance and recessiveness. Genetic analysis of 12 representative suppressors resulted in the assignment of these suppressors to 6 different loci, including the three previously described loci SUP35 (chromosome IV), SUP45 (chromosome II) and SUP46 (chromosome II), as well as three new loci SUP42 (chromosome IV), SUP43 (chromosome XV) and SUP44 (chromosome VII). Suppressors belonging to the same locus had a wide range of different phenotypes. Differences between alleles of the same locus and similarities between alleles of different loci suggest that the omnipotent suppressors encode proteins that effect different functions and that altered forms of each of the proteins can effect the same function.

scholarly journals From Structure to Sequence: Identification of polyclonal antibody families using cryoEM

2021 ◽  
Author(s):  
Aleksandar Antanasijevic ◽  
Charles A. Bowman ◽  
Robert N. Kirchdoerfer ◽  
Cristopher A. Cottrell ◽  
Gabriel Ozorowski ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Responses ◽  
Polyclonal Antibodies ◽  
Isolation And Characterization ◽  
Density Maps ◽  
Sequence Identification ◽  
Complementarity Determining Regions ◽  
Rate Limiting ◽  
Bioinformatic Approach

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity- determining regions and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next generation sequencing of immune repertoires we were able to specifically identify clonal family members, synthesize the monoclonal antibodies and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

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